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Fecal Contamination and Diarrheal Pathogens on Surfaces and in Soils among Tanzanian Households with and without Improved Sanitation Amy J. Pickering,∥,*,†,‡ Timothy R. Julian,∥,*,§ Sara J. Marks,† Mia C. Mattioli,† Alexandria B. Boehm,† Kellogg J. Schwab,§ and Jennifer Davis†,‡ †

Environment and Water Studies, Civil and Environmental Engineering, Stanford University, Stanford, California 94305, United States ‡ Woods Institute for the Environment, Stanford University, Stanford, California 94305, United States § Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland 21231, United States S Supporting Information *

ABSTRACT: Little is known about the extent or pattern of environmental fecal contamination among households using lowcost, on-site sanitation facilities, or what role environmental contamination plays in the transmission of diarrheal disease. A microbial survey of fecal contamination and selected diarrheal pathogens in soil (n = 200), surface (n = 120), and produce samples (n = 24) was conducted in peri-urban Bagamoyo, Tanzania, among 20 households using private pit latrines. All samples were analyzed for E. coli and enterococci. A subset was analyzed for enterovirus, rotavirus, norovirus GI, norovirus GII, diarrheagenic E. coli, and general and human-specific Bacteroidales fecal markers using molecular methods. Soil collected from the house floor had significantly higher concentrations of E. coli and enterococci than soil collected from the latrine floor. There was no significant difference in fecal indicator bacteria levels between households using pit latrines with a concrete slab (improved sanitation) versus those without a slab. These findings imply that the presence of a concrete slab does not affect the level of fecal contamination in the household environment in this setting. Human Bacteroidales, pathogenic E. coli, enterovirus, and rotavirus genes were detected in soil samples, suggesting that soil should be given more attention as a transmission pathway of diarrheal illness in low-income countries.

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INTRODUCTION Millennium Development Goal (MDG) Target 10 calls for halving, by 2015, the proportion of the global population without sustainable access to sanitation.1 To track progress toward MDG Target 10, the World Health Organization and UNICEF Joint Monitoring Programme for Water Supply and Sanitation (JMP) defines improved sanitation as access to a private toilet that is connected to a sewer or septic tank, a pourflush or ventilated improved pit latrine, a composting pit latrine, or a pit latrine with a concrete slab. In 2008, only 31% of the population in sub-Saharan Africa was classified as using an improved sanitation facility.2 The majority (90%) of sanitation facilities (both improved and unimproved) in sub-Saharan Africa are on-site pit latrines (Demographic and Health Surveys, data available at www.measuredhs.com). The presence or absence of a concrete slab thus typically determines whether a household has access to improved sanitation as defined by the JMP. Sanitation infrastructure interventions have been shown to reduce the incidence of diarrhea by 32−37%.3,4 In particular, JMP-defined improved sanitation facilities have been found to © 2012 American Chemical Society

be more strongly associated with lower child mortality and lower diarrhea than unimproved facilities.5 The association between sanitation and health is particularly relevant for subSaharan Africa, where diarrheal diseases are a leading cause of under-five child mortality and morbidity.6 There is evidence that level of sanitation is also linked to malnutrition and child growth.7,8 One hypothesized mechanism is the development of environmental enteropathy, a subclinical disorder characterized by intestinal malabsorption that may result from frequent ingestion of fecal bacteria.9,10 Improved sanitation has the potential to prevent environmental fecal contamination; however, it is largely unknown which features of a sanitation facility (e.g., concrete slab, roof, ventilation pipe, water seal) are important for preventing exposure to fecal bacteria and diarrheal pathogens.3,4 Received: Revised: Accepted: Published: 5736

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of soil collection, the sampling location was noted as “shaded”, “partly shaded”, or in “direct sunlight”. Bacteria were eluted from the soil using a method modified from Boehm et al.12 Approximately 5 (4.75−5.25) g of soil were eluted in 50 mL of distilled water in a 1 L Whirl-Pak bag (Nasco, Fork Atkinson, WI). The soil and water was shaken by hand for 2 min, then allowed to settle for 15 min. Twenty milliliters of supernatant was aliquoted into a sterile 50 mL polypropylene centrifuge tube. The supernatant was mixed by hand, and volumes of 1 and 5 mL were processed using membrane filtration for E. coli (EC) and enterococci (ENT) using EPA methods 1604 and 1600, respectively.13,14 All samples were processed within six hours of collection. For every 10 samples, a lab blank of distilled water was processed as a negative control and a duplicate volume was filtered from the same sample. The detection range of the assay is 2−5000 colony forming units (CFU) per gram of wet soil. Soil moisture content was determined using a microwave method adapted from Colorado State University Extension,15 as described in the SI. All bacterial concentrations are reported as log10 (CFU)/g dry weight. Virus was recovered from a subset of soil samples using a method modified from Dineen et al.16 Samples were chosen from five locations (house entrance, food preparation area, latrine entrance, latrine depth, and water activities area) at eight households (four with and four without a concrete slab), for a total of 40 samples. Approximately 35 (range 34−36) g of soil was added to 350 mL of distilled water in a 1 L Whirl-Pak bag. The soil and water were briefly shaken by hand and incubated for 1 min at room temperature. An aliquot from the sample was used to measure pH (Fisher Science Education pH Meter, Thermo Fisher Scientific Inc., Beverly, MA); if necessary, the sample was adjusted to pH 9−10 using 0.25 M NaOH. The sample was shaken by hand for 3 min then allowed to settle for 15−20 min. Two-hundred (200) mL of the supernatant was aliquoted into a 1 L Whirl-Pak bag. Four (4) mL of 2.5 M MgCl2 was added to the supernatant to achieve a target concentration of approximately 0.05 M MgCl2, and the pH was adjusted to 3−4 using 0.50 M HCl. Seventy (70) mL was filtered through a 0.45 μM pore size HA filter (Millipore, Billerica, MA). The filter was treated with 0.5 mL of RNALater (Qiagen, Valencia, CA), incubated at room temperature for 3 min, stored in a 5 mL centrifuge tube at −80 °C for 2−12 days, transported at room temperature for 2 days, and then stored at −80 °C until analysis. For every 20 samples, distilled water was processed as a negative control for virus assays. Surface Sampling. At each house a total of six fomites were sampled: a plastic plate, plastic cup, plastic washbasin, wooden broom handle, child’s toy, and the latrine wall (typically a plastic tarp). Bacteria were recovered from surfaces using polyester tipped swabs with wooden handles following a method modified from Moore et al.17 Each swab was vortexed in a 15 mL polypropylene centrifuge tube (BD Biosciences, Bedford, MA) containing 12 mL of 1/4 strength Ringer’s solution (Oxoid Ltd., Cambridge, UK) for 30 s. A volume of 5 mL was processed by membrane filtration within six hours of sample collection following standard EPA methods to enumerate EC and ENT.13,14 For every 12 fomite samples collected, a sterile swab was wetted in Ringer’s solution and similarly processed as a negative control for the bacterial fomite assay. The lower and upper limits of detection for the assay are approximately 2.4 and 1200 CFU per 100 cm2 surface, respectively.

Understanding fecal-oral disease transmission routes in lowincome countries has largely focused on pathogen transmission through drinking water, hands, and food. The role of fecal contamination on environmental surfaces as an exposure route for diarrheal disease has been relatively unstudied. Previous research conducted among households using on-site pit latrines in Tanzania has shown that, following handwashing, mothers’ hands become quickly recontaminated with fecal bacteria after touching environmental surfaces during household activities such as sweeping.11 This finding suggests that environmental fecal contamination is widespread; however, little is known about the extent or pattern of environmental fecal contamination among households using low-cost, on-site sanitation facilities. Indeed, few studies have assessed fecal contamination (e.g., fecal indicator bacteria, diarrheal pathogens) in soil or on surface samples from household environments in low-income countries. This study characterizes levels of fecal microorganisms in soils and on fomites (i.e., inanimate objects such as cups or toys capable of transmitting pathogens) among households in Tanzania with private pit latrines. Spatial patterns of fecal indicator bacteria (FIB) contamination in soil are examined with respect to latrine location and areas where common household activities are conducted. A subset of soil and surface samples is also analyzed for the presence of diarrheal pathogens, including gastrointestinal viruses (enterovirus, rotavirus, norovirus GI, and norovirus GII), diarrheagenic Escherichia coli, and general and human-specific Bacteroidales, using molecular methods. Finally, the association between presence of improved sanitation, as defined by the presence of a concrete slab on a pit latrine, and environmental fecal bacteria levels is assessed.

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MATERIALS AND METHODS A microbial survey of environmental fecal contamination and selected diarrheal pathogens was conducted among 20 households in the town of Bagamoyo, Tanzania, during March 2010. One half (n = 10) of sample households had a private unimproved pit latrine with an earthen floor, and the other half had a private improved pit latrine fitted with a concrete slab. All households occupied single-family dwellings with earth floors, and all had latrine facilities for the exclusive use of their household. All households included at least one child under five years of age. Soil Sampling. Ten soil samples were obtained from each household, at least one at each of the following locations: latrine entrance, latrine floor, house entrance, house floor, midpoint between the latrine and the house entrance, food preparation area, a water washing area (i.e., for laundry and dishes), and an area where no obvious activities were performed. At latrines with a concrete slab, the latrine floor was sampled by collecting soil from on top of, or adjacent to, the concrete slab. All soil samples were collected using a metal spade disinfected with RNase Away (Invitrogen, Carlsbad, CA) to collect a subset of soil from a layer 10 cm by 10 cm square and approximately 1 cm thick. See the Supporting Information (SI) for details on use of RNase Away as a disinfectant. The soil was collected into a sterile 50 mL polypropylene centrifuge tube (Corning Incorporated, Corning, NY). A soil sample was also obtained from a depth of 10 cm at the house entrance and the latrine entrance; the soil was collected from the bottom of a hole (10 × 10 × 10 cm) hand dug with the spade. At the time 5737

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Genomic DNA/RNA Extraction. Filters collected for virus analysis were assayed for the presence of four gastrointestinal viruses: enterovirus, norovirus GI, norovirus GII, and rotavirus. Soil samples were also assayed for the presence of general Bacteroidales and human-specific Bacteriodales. Viral RNA was extracted from surface samples using a modified acid guanidinium thiocyanate-phenol-chloroform extraction.26 A negative control was processed alongside each group of 12 samples during extraction of surface samples. For the 36 samples, this included two extraction blanks (distilled water) and one field blank (a swab wetted in Ringer’s solution and processed in the field during sample collection). Total DNA and RNA was extracted from soil samples using a modification of the IT 1−2−3 Platinum Path Sample Purification Kit (Idaho Technology Inc., Salt Lake City, UT). The negative controls included two extraction blanks (distilled water) and two field blanks (distilled water processed in a manner similar to soil samples and described above) for the 44 samples. Additional details on the extraction methods are provided in the SI. Detection of Genomic RNA for Gastrointestinal Virus. Virus genomic RNA was detected in soil samples using endpoint reverse transcription polymerase chain reaction (RTPCR) on a StepOnePlus RealTime PCR System (Applied Biosystems, Carlsbad, CA). Hepatitis G RNA was used to detect inhibition of RT-PCR reactions as described in the SI following the methods of Gibson et al.27 The reagents for enterovirus, hepatitis G, norovirus GI, norovirus GII, and rotavirus were used according to the manufacturer’s instructions for the AgPath-ID OneStep RT-PCR Mastermix Kit (Applied Biosystems, Inc., Foster City, CA) for a 25 μL reaction with 5 μL template; five U of RNase Inhibitor (Applied Biosystems) was also added to the Mastermix. The primers and probes for enterovirus, hepatitis G, norovirus GI, norovirus GII, and rotavirus are presented in SI Table S1.28−31 The cycling conditions for enterovirus, hepatitis G, norovirus GI, and norovirus GII were according to the Mastermix manufacturer’s instructions; the cycling conditions for rotavirus are provided in the SI.29 Negative controls were included during the RT-PCR for each target virus in the form of no template controls. Positive controls, as described in the SI, were included for each target virus with every RT-PCR run. A sample was considered positive if the cycle threshold (Ct) was less than 40, consistent with the limit of detection determined by diluting positive controls to extinction (data not shown). The estimated lower limit of detection (LLOD) for the surface virus assay (collection, preservation, extraction, and detection) is 80 000 genomic copies per 100 cm2. The estimated LLOD for the soil virus assay is approximately 150 genomic copies per gram. See SI for further details. Detection of Genomic DNA for General and Humanspecific Bacteroidales. Soil samples from eight households (four with and four without a concrete slab) were also assayed for presence of general and human-specific Bacteroidales genomic DNA using quantitative polymerase chain reaction (qPCR) on a StepOnePlus RealTime PCR System (ABI, Foster City, CA). The reagents and cycling conditions used were according to the manufacturer’s instructions for the TaqMan Universal PCR Mastermix (ABI) for a 25 μL reaction with 5 μL template; bovine serum albumin (ABI) was also added to a final concentration of 0.2 mg/mL.32,33 The primers and probes for general and human-specific Bacteroidales were developed for

For a subset of samples, a second swab was collected from a different 100 cm2 area of the same, or similar, fomite to assay for presence of gastrointestinal virus. All six fomites were tested from six houses (36 samples total). Swabs were processed by pouring the full volume (12 mL) of sample onto an HA filter housed in a membrane filtration apparatus containing approximately 10 mL of 0.10 M MgCl2, adjusted to pH 3.0 using 0.5 M HCl, incubated at room temperature for 1 min, and then vacuum filtered. The filter was treated with 0.5 mL of RNALater (Qiagen, Valencia, CA), incubated at room temperature for 3 min, stored in a 5 mL centrifuge tube at −80 °C for 2−12 days, transported at room temperature for 2 days, and then stored at −80 °C until analysis. For every 18 samples, distilled water was processed as a negative control for the virus assays. Produce. Fresh produce was sampled at households when available, and from three local produce vendors. Items sampled included mango, tomato, onion, cabbage, potato, pepper, lime, sugar cane, African plum, and beans. Each piece of produce was rinsed in 350 mL of distilled water in a sterile Whirl-Pak bag for 30 s and massaged through the bag. Volumes of 1 and 10 mL were membrane filtered to enumerate EC and ENT using EPA methods; the detection limit ranged from 35 to 175 000 CFU per item. E. coli Pathotype Gene DNA Extraction and PCR. Following incubation, membrane filters with E. coli biomass were lifted from the MI agar and archived for detection of pathotype genes for five diarrheagenic E. coli pathotypes, including enteroinvasive (EIEC), enteropathogenic (EPEC), enteroaggregative (EAEC), enterotoxigenic (ETEC), and enterohemorrhagic (EHEC). Bacterial DNA was extracted from archived filters collected from six households using solutions and beads provided by the MoBio PowerSoil Kit (MoBio Laboratories, Inc., Carlsbad, CA) following the protocol described in Pickering et al.11 A total of 47 soil samples and 17 surface samples were analyzed by multiplex conventional PCR. Samples that were below the limit of detection of the culture-based E. coli assay (no growth on MI filters) were not archived or processed for pathotype genes. In addition, eight produce samples were processed (two tomatoes, two mangos, two onions, and two potatoes). For each type of produce, one was collected from a sample household and one was collected from a local produce vendor. Since both total coliform and E. coli grow on MI agar, archived filters may have contained biomass from total coliform in addition to E. coli. Three multiplex PCR assays were run on each sample using reagents supplied in the Type-It Mutation Detect PCR Kit (Qiagen, Valencia, CA). Two microliters of each DNA extract were used as template in each 25 μL multiplex PCR reaction. The reaction composition included 1X Type-It Multiplex PCR Master Mix (2X), 0.5X Q-Solution (5X), 1X CoralLoad Dye (10X), and primers at concentrations listed in SI Table S1. Multiplex 1 targeted the aggR gene of EAEC and the eaeA gene of EPEC,18−20 multiplex 2 targeted the stx1 and stx2 genes of EHEC, and the ipaH gene of EIEC,21,22 and multiplex 3 targeted the Lt1 and st1b genes of ETEC.20,23−25 Thermocycler conditions are detailed in the SI. A positive result was recorded when a PCR product of appropriate size (SI Table S1) was observed following gel electrophoresis. Positive PCR amplicons from one to three samples per target were sequenced to confirm amplification of the intended product (see SI for details). 5738

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EPA Method B in Haugland et al. and Seifring et al., as presented in SI Table S1.32,33 Standard curves were generated for general and humanspecific Bacteroidales markers to quantify concentrations. Standards were generated using whole genomic DNA from B. thetaiotaomicron for general Bacteroidales and using sequencespecific plasmid DNA from an E. coli clone harboring the Bacteroidales human-specific rRNA gene for human-specific Bacteroidales, as previously described in Walters et al. (2009).34 Further details of standard curves are in the SI. For the general and human-specific Bacteroidales assays, the LLOD are 6.7 gene copies (1.1 cell equivalents) and 5.2 gene copies (5.2 cell equivalents) per gram soil, respectively; the lower limits of quantification (LLOQ) are 67 gene copies (11 cell equivalents) and 52 gene copies (52 cell equivalents), respectively, per gram soil. When general and human-specific Bacteroidales was not detected in a sample, the sample was assigned a concentration of 1 cell equivalent per gram soil for all analyses; the geometric mean of the range between LLOD and LLOQ (3.3 cell equivalents for general Bacteroidales and 16 cell equivalents for human-specific Bacteroidales) was used when Bacteroidales was detected at concentrations below the LLOQ. Cell equivalents were log10 transformed for statistical analysis. Inhibition of the general Bacteroidales assay is discussed in the SI. Data analysis. Data were analyzed using IBM SPSS Statistics 19 (IBM Company, Armonk, NY). One-way ANOVA with post hoc comparisons (Tukey HSD) and independent sample t tests were used, as described in the SI. Results were considered statistically significant at a significance level of α ≤ 0.05.

Figure 1. Log-mean colony forming units (CFU) of E. coli (EC) and enterococci (ENT) per gram of soil by sampling location. “Midpoint” refers to the midpoint between the house entrance and the latrine entrance. “No activities” refers to an area of the plot that appeared not to be used regularly. Depth samples were obtained by digging 10 cm below the soil surface. Error bars display standard error of the mean.

surface (mean per gram soil: 0.5 log10 CFU EC; 1.1 log10 CFU ENT) were significantly lower than all other sampling locations (all post hoc comparisons p < 0.05). Soil collected from areas with no apparent household activities had significantly lower concentrations of EC (mean 1.8 log10 CFU) and ENT (mean 2.3 log10 CFU) per gram of soil than the house floor and food preparation area (p < 0.05). The midpoint between the house and the latrine had significantly lower levels of bacteria (mean 1.9 log10 CFU EC; 2.1 log10 CFU ENT) than the house entrance and house floor (all p < 0.05). FIB concentrations in soil were higher in shaded areas (Figure 2); samples collected from locations in direct sunlight

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RESULTS Household Characteristics. Participating households had an average of seven members. Direct observation confirmed that each household had access to one pit latrine within the household plot. None of the latrines was fitted to a ventilation pipe, had a water seal, or was connected to a septic tank. Of all 20 latrines, three (15%) had a roof, 12 (60%) had a pit cover at the time of data collection, and 12 (60%) had visible water or urine around the pit. Soap was observed in only two (10%) latrines; water in a container was present in nine (45%) latrines. Additionally, 15 (75%) latrines had visible flies, and eight (40%) had visible live maggots in the pit. The average measured walking distance from the latrine entrance to the household entrance was 9.5 m (SD 4 m, range 4−21 m). In each cohort, 7 out of 10 households had only one child under five; the rest had two children under five. Soil. Soil samples had a mean of 2.1 log EC (SD 1.4) and 2.5 log ENT (SD 1.1) per gram of soil. There were no statistically significant differences in soil FIB levels between households using a pit latrine with and without a concrete slab (p > 0.05, SI Table S2). The concentration of EC and ENT found in soil differed significantly by sampling location (one-way ANOVA, p < 0.001), but did not differ significantly by household (one-way ANOVA, p > 0.05). Figure 1 shows log-mean EC and ENT concentrations per dry gram of soil by sampling location. Post hoc tests revealed that the house floor had an average of 1.3 log10 CFU more EC (p = 0.010) and 0.9 log10 CFU more ENT (p = 0.035) per gram of soil as compared to the latrine floor. The highest bacterial concentrations were found in soil collected from the house floor, house entrance, food preparation area, and water activities area. The levels of bacteria in soil samples collected 10 cm below the ground

Figure 2. Log-mean CFU of E. coli (EC) and enterococci (ENT) per gram of soil by level of sunlight at time of sampling. Depth soil samples not included. Error bars show the standard error of the mean.

had significantly lower levels of bacteria (EC: t=-3.4, df=131, p = 0.001; ENT: t = −3.6, df = 132, p < 0.001). In addition, soil moisture content was positively correlated with log CFU EC (rp = 0.15, p = 0.041) and log CFU ENT (rp = 0.14, p = 0.054) per gram of soil. E. coli pathotype genes were detected in soil from all sampling locations at least once, and a total of 72% of the samples analyzed tested positive for at least one E. coli pathotype gene (Table 1, SI Table S3). Notably, 100% of 5739

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Table 1. Percentage and Number of Soil, Surface, and Produce Samples Positive for Pathotype Genes of Diarrheagenic E. coli (DEC)a % (N)

EAEC

EPEC

EHEC

EIEC

ETEC

any DEC

soil (n = 47) surface (n = 17) produce (n = 8)

11% (5) 0% (0) 13% (1)

17% (8) 6% (1) 13% (1)

23% (11) 47% (8) 25% (2)

51% (24) 47% (8) 38% (3)

28% (13) 35% (6) 13% (1)

72% (34) 77% (13) 50% (4)

a

Including enteroinvasive (EIEC), enteropathogenic (EPEC), enteroaggregative (EAEC), enterotoxigenic (ETEC), and enterohemorrhagic (EHEC).

detection limit for the general Bacteroidales marker than all other locations (p < 0.001). There was no significant difference between cell equivalents per gram of general Bacteroidales found in soil samples collected from households with or without a concrete latrine slab (p = 0.3). Further discussion of inhibition of the general Bacteroidales is available in the SI. Surfaces. The mean level of ENT (1.0 log10 CFU/100 cm2, SD 1.0) found on surfaces was 0.5 log10 CFU higher per 100 cm2 than the mean level of EC (0.5 log10 CFU/100 cm2, SD 0.7). The highest levels of ENT and EC were found on plastic plates and cups; the lowest levels were found on the latrine wall (Figure 4). Surfaces that were observed to be visibly wet had an

samples taken from the water activities area tested positive for E. coli pathotype genes; the majority of samples taken from the house floor (83%), no apparent activities (83%), food preparation (67%), and path between the house and latrine (67%) were also positive. Enterovirus and rotavirus gRNA were detected in 2 (5%) and 1 (2.5%) of the 40 soil samples, respectively. The positive samples were all obtained from a single household, at the house entrance (enterovirus and rotavirus) and the food preparation area (enterovirus). The household latrine had a concrete slab. Norovirus (GI or GII) was not detected in any of the soil samples tested. The samples were minimally impacted by RTPCR assay inhibition, as demonstrated by a ΔCt of spiked hepatitis G gRNA of less than 3.3 for most samples (31 of 44 total samples). Diluting the inhibited samples 10-fold in RNasefree water reduced inhibition as demonstrated by hepatitis G RNA signal ΔCt < 3.3 in the diluted samples, but results for gastrointestinal viruses remained unchanged. General and human-specific Bacteroidales were detected in 28 (70%) and 7 (18%) of the 40 soil samples tested, respectively. Figure 3 shows log-mean Bacteroidales concentrations in soil by

Figure 4. Mean log colony forming units (CFU) of E. coli (EC) and enterococci (ENT) per 100 cm2 of surface sampling location. Error bars represent the standard error of the mean.

average of 0.3 log CFU more EC per 100 cm2 (p = 0.046) and 0.6 log CFU ENT per 100 cm2 (p = 0.003) compared to dry surfaces. Concentrations of EC or ENT on surfaces with visible dirt were not significantly different than concentrations on visibly clean surfaces (p > 0.05). Approximately three-fourths (77%) of surface samples analyzed tested positive for an E. coli pathotype gene. E. coli pathotype genes were found on all surface sampling locations except for the latrine wall (Table 1, SI Table S4). Of all surface samples that tested positive for an E. coli pathotype gene, most (62%) were from cups and plates. Only one sample was positive from a wooden broom handle, and only one from a plastic water basin. No virus gRNA was detected on any of the fomite samples tested (N = 36); there was minimal RT-PCR assay inhibition (ΔCt < 3.3 for spiked hepatitis G RNA). Produce. Among all produce tested, items from vendors did not have statistically different levels of fecal bacteria compared to items from households (p > 0.05). Produce had a mean of 3.4 log CFU E. coli (SD 1.8) and mean of 4.8 log CFU ENT (SD 0.9) per item (Figure S1). Four produce items (50%) were

Figure 3. Log-mean cell equivalents of general and human-specific Bacteroidales detected in soil samples in five locations from eight households. Error bars represent the standard error of the mean. “ND” is defined as no Bacteroidales detected in any of the eight samples tested.

sampling location. Of the seven samples positive for humanspecific Bacteroidales, two were above the LLOQ. Concentration of general Bacteroidales found in soil differed significantly by sampling location (one-way ANOVA, p < 0.001), but not by household (one way ANOVA, p = 0.587). Post hoc tests revealed that Bacteriodales was an average of 1.4 log10 cell equivalents per gram soil higher in the house entrance than both the latrine entrance (p = 0.026) and the water activities area (p = 0.027). Soil samples collected 10 cm below the surface were significantly more likely to be below the 5740

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positive for at least one E. coli pathotype, including two mangos, one tomato, and one onion (Table 1). The tomato and onion were positive for only one E. coli pathotype. Both of the mangos were positive for three E. coli pathotypes. Direct comparison between produce items is complicated by the fact that surface area and surface texture varies between items. Blanks and Sequencing Data. All field and extraction blanks for EC, ENT, E. coli pathotype genes, general and human-specific Bacteroidales and gastrointestinal virus gRNA were negative. We obtained good and fair quality sequence reads for ETEC LT1, ETEC st1b, EHEC stx1, EHEC stx2, EAEC aggR, and EPEC eaeA from one to three samples. However, we did not obtain a good quality sequence read for EIEC ipaH. The sequences that had good and fair reads aligned with their intended targets in GenBank with between 91 and 99% maximum identity values (SI Table S5).

among all soil sampling locations. Surfaces that were observed to be visibly wet were also found to have higher levels of EC and ENT. Considering that 85% of latrines did not have a roof, soil exposure to sunlight may have contributed to reducing levels of FIB on the latrine floor and entrance. Previous work in Hawaii and Guam suggests that EC and ENT may be indigenous soil bacteria in the tropics;37,39 however, there are several indications that the microbial contamination detected in this study is from fecal sources. In addition to EC and ENT, we detected general and humanspecific Bacteroidales, pathogenic E. coli genes, and viral genomes. Both general and human-specific Bacteroidales have low potential for regrowth in the environment, and the humanspecific Bacteroidales HF183 assay has demonstrated very high specificity to human hosts.32,40−42 Additionally, concentrations of FIB were significantly lower in the 10 cm deep soil samples than at the soil surface; presumably, autochthonous soil FIB would have been found throughout the soil. It should be noted that FIB and general Bacteroidales detected could possibly be from chicken or other livestock feces; even if this is the case, animal feces can contain human pathogens such as Cryptosporidium and Giardia.43,44 Further microbial source tracking to identify the precise sources of fecal contamination in settings like this is warranted. The study has some notable limitations. Although 10% of samples were processed in duplicate at the assay level, there were no duplicate samples processed at the sample collection level. Quantifying within-sample variability would provide a better understanding of the findings that fecal contamination is significantly associated with location within a household. Furthermore, the limited sample size (20 houses) prevented analysis including potential control variables that may have impacted levels of environmental contamination (e.g., number of children under five, distance to latrine). Importantly, sunlight and moisture content were not controlled for in this study. Although these environmental factors were found to be associated with bacteria contamination in soils, future research with a randomized study design is needed to support these findings. The molecular markers used in this study are highly specific to the microbial targets (enterovirus, rotavirus, general Bacteroidales, human-specific Bacteroidales, and E. coli pathotype genes); however, their detection does not necessarily indicate presence of infective pathogens. The E. coli pathotype genes were detected in DNA obtained from membrane filters containing biomass of E. coli and other coliforms. Thus we cannot rule out the possibility that the pathotype genes were in other coliform, and not E. coli. The detection of these genes remains important, as E. coli pathotype genes such as stx1 and stx2 have been documented to be capable of horizontal gene transfer.45 We sequenced a subset of the amplicons from the E. coli pathotype gene assays and found the sequences aligned well with their intended gene target (SI Table S5). Future studies investigating environmental contamination of pathogenic E. coli should employ further molecular characterization for confirmation. The fecal contamination levels measured in soil, on surfaces, and on produce are also not necessarily indicative of infection risks. For example, produce preparation influences an individual’s exposure to surface bacteria during consumption.46 This study provides new evidence that diarrheagenic bacteria and enteric viruses exist in soil and on fomites in Africa. The presence of FIB, E. coli pathotype genes, rotavirus, and enterovirus in the food preparation area and inside the home

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DISCUSSION High levels of EC and ENT detected in soil and on fomites indicate that environmental fecal contamination is pervasive in Tanzanian households that use pit latrines. Concentrations of FIB and general Bacteroidales were higher in soil samples collected from inside the house and food preparation areas compared to soil collected near or inside the latrine. It is notable that fomite sampling in the U.S. shows a similar trend despite the different setting: higher levels of microbial contamination are found in kitchens than in bathrooms.35 One potential explanation for this trend in Tanzania is that the latrine may not be the primary source of fecal contamination among study households. Children defecating directly on the ground, and/or wastewater disposal in the yard, could contribute to fecal contamination near the house.36 It is also important to acknowledge that FIB may be indigenous soil bacteria in the tropics,37 and that off-plot sources of FIB are possible. For example, produce sampled in markets and households was found to have high levels of fecal bacteria and to carry E. coli pathotype genes. The potential for produce to transport fecal contamination into households is consistent with previous research demonstrating that preparing food increases fecal bacteria levels on mothers’ hands.11 No significant association was found between soil fecal bacteria levels and the presence of a concrete slab in the latrine. This result suggests that a concrete slab does not prevent the spread of fecal contamination within the household environment, and/or that the latrine is not the main source of soil and surface fecal contamination in this setting. Additional research is necessary to determine if there are specific latrine features significantly associated with reduced fecal contamination and pathogen presence in the home environment. Future work should also investigate levels of soil and fomite fecal contamination among households lacking access to on-site sanitation facilities. Similarly, there is a need for additional research investigating soil-transmitted pathogens not measured in this study (e.g., helminths), as they may behave differently than FIB and viruses with respect to environmental fate and transport. Sunlight and moisture may play important roles in influencing levels of contamination in soil and on surfaces, as has been suggested for beach sands in the U.S.38 Lower levels of FIB were found in soil located in direct sunlight, whereas higher levels were found in soils with higher moisture content. Our results indicate that the water activities area exhibited the highest log-mean concentration of CFU EC per gram soil 5741

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(5) Fink, G.; Gunther, I.; Hill, K. The effect of water and sanitation on child health: Evidence from the demographic and health surveys 1986−2007. Int. J. Epidemiol. 2011, 40 (5), 1196−1204. (6) Levels and Trends in Child Mortality; UNICEF, 2011. (7) Checkley, W.; Gilman, R. H.; Black, R. E.; Epstein, L. D.; Cabrera, L.; Sterling, C. R.; Moulton, L. H. Effect of water and sanitation on childhood health in a poor Peruvian peri-urban community. Lancet 2004, 363 (9403), 112−118. (8) Esrey, S. A. Water, waste, and well-being: A multicountry study. Am. J. Epidemiol. 1996, 143 (6), 608−623. (9) Humphrey, J. Child undernutrition, tropical enteropathy, toilets, and handwashing. Lancet 2009, 374, 1032−1035. (10) Lunn, P. The impact of infection and nutrition on gut function and growth in childhood. Proc. Nutr. Soc. 2000, 59, 147−154. (11) Pickering, A. J.; Julian, T. R.; Mamuya, S.; Boehm, A. B.; Davis, J. Bacterial hand contamination among Tanzanian mothers varies temporally and following household activities. Trop. Med. Int. Health 2011, 16 (2), 233−239. (12) Boehm, A. B.; Griffith, J.; McGee, C.; Edge, T. A.; SoloGabriele, H. M.; Whitman, R.; Cao, Y.; Getrich, M.; Jay, J. A.; Ferguson, D.; Goodwin, K. D.; Lee, C. M.; Madison, M.; Weisberg, S. B. Faecal indicator bacteria enumeration in beach sand: A comparison study of extraction methods in medium to coarse sands. J. Appl. Microbiol. 2009, 107 (5), 1740−1750. (13) USEPA. Method 1600: Enterococci in Water by Membrane Filtration Using Membrane-Enterococcus Indoxyl-D-Glucoside Agar (mEI), EPA 821/R-02/022; U.S. Environmental Protection Agency, Office of Water (4303T): Washington, DC, 2006. (14) USEPA. Method 1604: Total Coliforms and Escherichia Coli in Water by Membrane Filtration Using a Simultaneous Detection Technique (MI Medium), EPA-821-R-02-024; U.S. Environmental Protection Agency: Washington DC, 2002. (15) Schneekloth, J.; Bauder, T.; Broner, I.; Waskom, R. Measurement of soil moisture. In Colorado State University Cooperative Extension Drought and Fire Tip Sheets. www.ext.colostate.edu/ drought/soilmoist.html 2002 (accessed January 27, 2011). (16) Dineen, S. M.; Aranda, R.; Dietz, M. E.; Anders, D. L.; Robertson, J. M. Evaluation of commercial RNA extraction kits for the isolation of viral MS2 RNA from soil. J. Virol. Methods 2010, 168 (1), 44−50. (17) Moore, G.; Griffith, C. Problems associated with traditional hygiene swabbing: The need for in-house standardization. J. Appl. Microbiol. 2007, 103 (4), 1090−1103. (18) Ratchtrachenchai, O. A.; Subpasu, S.; Ito, K. Investigation on enteroaggregative Escherichia coli infection by multiplex PCR. Bull. Dep. Med. Sci. 1997, 39, 211−220. (19) Toma, C.; Lu, Y.; Higa, N.; Nakasone, N.; Chinen, I.; Baschkier, A.; Rivas, M.; Iwanaga, M. Multiplex PCR assay for identification of human diarrheagenic Escherichia coli. J. Clin. Microbiol. 2003, 41 (6), 2669. (20) Brandal, L. T.; Lindstedt, B. A.; Aas, L.; Stavnes, T. L.; Lassen, J.; Kapperud, G. Octaplex PCR and fluorescence-based capillary electrophoresis for identification of human diarrheagenic Escherichia coli and Shigella spp. J. Microbiol. Methods 2007, 68 (2), 331−341. (21) Brian, M.; Frosolono, M.; Murray, B.; Miranda, A.; Lopez, E.; Gomez, H.; Cleary, T. Polymerase chain reaction for diagnosis of enterohemorrhagic Escherichia coli infection and hemolytic-uremic syndrome. J. Clin. Microbiol. 1992, 30 (7), 1801−1806. (22) Vidal, R.; Vidal, M.; Lagos, R.; Levine, M.; Prado, V. Multiplex PCR for diagnosis of enteric infections associated with diarrheagenic Escherichia coli. J. Clin. Microbiol. 2004, 42 (4), 1787−1789. (23) Rappelli, P.; Maddau, G.; Mannu, F.; Colombo, M.; Fiori, P.; Cappuccinelli, P. Development of a set of multiplex PCR assays for the simultaneous identification of enterotoxigenic, enteropathogenic, enterohemorrhagic and enteroinvasive Escherichia coli. New Microbiol. 2001, 24 (1), 77. (24) Lopez-Saucedo, C.; Cerna, J. F.; Villegas-Sepulveda, N.; Thompson, R.; Velazquez, F. R.; Torres, J.; Tarr, P. I.; EstradaGarcia, T. Single multiplex polymerase chain reaction to detect diverse

suggest that soil is an important input of diarrheal pathogens to the fecal-oral route of illness transmission, one that has received little attention to date. These results highlight a need to understand better the source, distribution, and fate of fecal contamination in households, including fecal contamination transport between multiple environmental reservoirs (e.g., stored water, hands). Widespread contamination of the household environment with fecal bacteria could play a principal role in child exposure to diarrheal pathogens and fecal bacteria, considering that children under five are likely to have frequent hand-to-soil and hand-to-mouth contact.47 Future research should focus on the effect of exposure to contaminated soils and fomites on child health, including risk of diarrheal illness and environmental enteropathy. A next step will be to identify strategies that block diarrheal pathogen transmission through soil or fomites, such as changing household surfaces to materials that can be easily disinfected.

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ASSOCIATED CONTENT

S Supporting Information *

Detailed information on methods (soil moisture content, genomic DNA/RNA extraction and detection, sequencing), results (inhibition, influence of shade on FIB, detection of FIB and molecular markers), Tables S1−S5, and Figure S1. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (A.J.P.); [email protected] (T.R.J.). Phone: 510-410-2666 (A.J.P.); 650-241-8752 (T.R.J.). Author Contributions ∥

These authors contributed equally to this work

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

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REFERENCES

We thank Angela Rice, Michael Harris, Muhsin Boi, Lupakisyo Adam, participating households, and four anonymous reviewers. This study was supported by the National Science Foundation (SES-0827384), the Woods Institute for the Environment, and the Freeman Spogli Institute for International Studies at Stanford University, the Osprey Foundation of Maryland, Inc., and the Johns Hopkins University Global Water Program.

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